The purpose of these studies is to develop imaging techniques to monitor sub-cellular structures and processes, in vivo. We have been systematically developing an in vivo optical microscopy system that is adapted to biological tissues and structures rather than forcing an animal on a conventional microscope stage. This year we added the use of 3 dimensional electron microscopy and super optical resolution imaging to the technologies in the lab through strategic collaborations. The following major findings were made over the last year: 1) We published a compact design for conducting total emission detection (TED) on biological tissues in vivo. Using this device we were able to demonstrate 2 to 5 fold increased sensitivity in multiphoton excitation microscopy, in vivo. This prototype is being developed into a commercially available design. 2) Using Independent Component Analysis (ICA) we published the methods of increasing the signal to noise of fluorescence microscopy when more than one fluorescent probe is being utilized. This approach permitted the proper registration of the probes in the tissue with minimal spectral filtering greatly improving the efficiency of the process. 3) We completed a major study in characterizing the structure of mitochondria in skeletal muscle cells using 3D electron microscopy, histochemical and super resolution microscopy. These studies revealed the presence of a mitochondrial reticulum that we proposed distributes energy rapidly throughout the cell via the mitochondrial membrane potential rather than the slow diffusion of molecules. This is a novel concept in muscle energetics and has opened many new questions on its regulation, formation and presence in other tissues. We have completed our initial studies in heart tissue and found that though the mitochondrial reticulum is structured differently, it serves a similar role in the heart. The further evaluation and characterization of this structure in skeletal muscle and heart will be a major project in the next year.